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[EN] The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, beta nerve growth factor ((beta-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as beta-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and beta-NGF, they could be used to develop new extenders with less hormone concentration. (C) 2017 Elsevier Inc. All rights reserved., This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L. Casares-Crespo is supported by a scholarship from Instituto Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund. P. Fernandez-Serrano is supported by funds from Instituto Valenciano de Investigaciones Agrarias (IVIA) and Ministerio de Empleo y Seguridad Social (Programa de Garantia Juvenil).

[EN] Since ovulation in rabbit is induced by sensory stimulation associated with mating, an effective control of ovulation is required in the artificial insemination process. The intramuscular application of GnRH analogues is the most common practice in rabbit reproductive management; however, the process can be simplified replacing the intramuscular application by the addition of the GnRH analogue to the semen extender, thus reducing the time needed to carry out the insemination, the risks associated with the use of needles and animal distress. The efficiency of GnRH analogue when administered with the semen extender depends on the degradation of the hormone by aminopeptidases, a group of enzymes present in seminal plasma. In this final degree project, aminopeptidase activity present in seminal plasma of males belonged to the New Zealand White line from five months (beginning of the training of those males used in artificial insemination) to nine months old (adult males with a normal semen production) is determined. For this, semen samples from each male were collected weekly and aminopeptidase activity was measured using three different substrates: alanine, cysteine and leucine-β- naphthylamide. Regarding aminopeptidase activity, it can be distinguished a group of males with a high aminopeptidase activity (around 1000 pmol/mg protein/min) and another group with half of the activity approximately. Correlation between quality sperm parameters and aminopeptidase activity was found, concretely with acrosome status and sperm abnormalities. Significant differences for aminopeptidase activity and male age or kinetic sperm parameters were not found. Regarding the results, it can be concluded that aminopeptidase activity varies among males and is not dependent on male age; this new parameter could be used to choose the best males for the artificial insemination procedure which involves GnRH analogue application with the semen extender., [ES] En la inseminación artificial del conejo, al ser una especie de ovulación inducida, se requiere un control efectivo de la ovulación, por lo que en el momento de la inseminación hay que inducir la ovulación, lo que habitualmente se hace mediante la aplicación intramuscular de un análogo sintético de la GnRH. El proceso puede simplificarse sustituyendo la aplicación intramuscular de la GnRH mediante su incorporación en el diluyente, lo que reduce el tiempo de aplicación de la dosis seminal a la vez que evita el riesgo asociado al uso de jeringuillas del personal que aplica la dosis y el estrés animal asociado al pinchazo. La eficacia de la hormona en el diluyente depende de varios factores, entre los que destaca la degradación del análogo por parte de las aminopeptidasas, unas enzimas presentes en el plasma seminal. En el presente Proyecto de Fin de Grado se determinó la actividad aminopeptidasa presente en el plasma seminal en machos de origen neozelandés desde los cinco meses de edad (inicio del entrenamiento de los machos utilizados en inseminación artificial) hasta los nueve meses de edad (machos adultos en plena producción de semen). Para ello, se recuperaron muestras de semen semanalmente de los machos y se determinó la actividad aminopeptidasa usando tres sustratos diferentes: alanina, cisteína y leucina-β-naftilamida. En base a los resultados obtenidos, se distingue un grupo de machos con alta actividad aminopeptidasa (alrededor de 1000 pmol/mg protein/min) y otro con una actividad de aproximadamente la mitad. Se observó cierta correlación entre algunos parámetros de la calidad del semen y la actividad aminopeptidasa, concretamente con el estado del acrosoma y anormalidades en el esperma. La edad de los machos no afectó significativamente a la actividad aminopeptidasa. Con los resultados obtenidos se puede concluir que la actividad aminopeptidasa varía en función del macho y es independiente de su edad, pudiéndose utilizar este nuevo parámetro para seleccionar los mejores machos para la técnica de inseminación artificial que utiliza GnRH en el diluyente.

[EN] Ovulation induction in artificially inseminated rabbits by adding GnRH synthetic analogues in the seminal doses is a welfare-orientated method to induce ovulation in rabbits and could have some advantages in field practice. This study was conducted to determine the effect of male genotype on the aminopeptidase activity in rabbit seminal plasma and the effects of dilution rate of semen on availability and reproductive performance when buserelin acetate is added to the seminal dose. To study the aminopeptidase activity, 12 mature bucks belonging to a paternal line and 12 from a maternal line were used. The bucks from the paternal line were used to study the effect of dilution rate on the availability of buserelin acetate after 2 hours of dilution and on the reproductive performance of the doses after artificial insemination of 389 commercial crossbreed does. Aminopeptidase activity in seminal plasma is dependent on the male genotype. The paternal line resulted 27% more aminopeptidase activity than the maternal line (P < 0.05). On the other hand, semen diluted 1:20 exhibited a marked increase in the availability of buserelin acetate and the fertility in this group was significantly higher than females from dilution rate 1:5 group, which showed similar results to that of the negative control group (does inseminated with semen diluted 1:20 in non-GnRH-supplemented extender). We conclude that the bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases and is consequently affected by the dilution rate used to prepare the artificial insemination doses. (C) 2014 Elsevier Inc. All rights reserved., This research was supported in part by the RTA2010-00117-00-00 from INIA and the European FEDER Funds. E. Moce is supported by funds from Ministerio de Ciencia e Innovacion from Subprograma Ramon y Cajal and the European Social Fund (ref. RYC-2010-06162; Madrid, Spain).